Lewy Bodies in PD.Lewy Body Inclusions and the effect of coffee and decaffeinated extracts on Drosophila neuronal cultures

Introduction to PD• Parkinson’s disease is a

chronic neurodegenerative disease characterized by a variety of motor and non-motor symptoms with no proven successful therapy.

• Typical hallmarks of PD are:1. Loss of Dopaminergic

neurons in the Substantia nigra pars compacta region of the brain leading to motor disorders.

2. Presence of LB in the surviving Dopaminergic Neurons.

Two Types of Parkinson’s disease• Sporadic PD: Non-

inheritable, onset due to chemical insults resulting in mutations in associated genes such as LRRK2 or SNCA genes

• Familial PD: Mainly associates to Autosomal dominant mutations in genes such as SNCA.

• A53T: increases propensity of Fibrillar LB formations

• E46K: increases Propensity of Protofibrils or Oligomeric a-syn to form

• A30P: accelerates protofibrillar formation.

What is Alpha-synuclein• Alpha synuclein is the culprit of

LB formation• It is:

1. 140 amino acid protein. The α-syn has a 12-amino acid stretch in the hydrophobic core and is concerned with the fibrillar aggregation in DA cells.

2. Natively unfolded protein with a hydrophobic region and an acidic tail as is the same with beta and gamma synuclein.

3. Believed to be extensively localized in the nucleus and found in the synapse thus the name synuclein. But its function is still under study.

Mark R. Cookson, The Biochemistry of Parkinson’s disease

What is LB from other science journals• Lewy bodies are fibrillar

protein aggregations found in surviving dopaminergic neurons in PD.

• Its main constituents of interest are α-syn and ubiquitin and β amyloid fibrils.

• LB forms from Oligomeric α-syn which arises from monomeric α-syn.

• Some papers say the oligomeric form is toxic which disrupts cellular permeability and LB is a cellular rescue response; others, LB aggregations trigger aptosis.

• Even though LB are associated with fibrils which can be stained with immunogold, However, it has been tested that A-53T mutant form is able to form aggregates by it self

Drasophila Melanogaster• Why Drasophila

melanogaster:1. Cheap and easy to

maintain2. High reproduction rate3. Entire genome has been

sequenced4. Readily manipulate gene

expression in the embryo creating gain or loss-of-function conditions almost at will.

A53T line is used because the mutation accelerates LB aggregation

Antibodies and dyes• Experiments have been

done to show that the 1:5000 of primary antibodies give the best distinction between LB and background signals

• DAPI: stains the nucleus (Blue) (1:2000)

• Anti-HRP: stains the Na+K+ channels on cells membrane (Green) (1:100)

• Primary antibodies: selectively stain for a-syn (1:5000)

• Secondary antibodies: selectively binds to primary antibodies and emits orange light. (1:2000)

0.000

1.000

2.000

3.000

4.000

5.000

6.000

5.051

3.8873.519 1 in 5000

1 in 75001 in 10,000

LB

/BG

Inte

nsi

ty

Objectives:

•Define Lewy Bodies•Quantify the number of LB in A53T cell

line over 1.2.3.6 and 9 day-in-vitro (DIV) and total number of DA cells in the same culture.

•Determine if LB is a cause or a rescue response of PD.

Methodology:• Eggling of A53T fruit flies for 4½ to 5 hours.• Cells are extracted from the eggs at the right stage.• Cells are grown in special mediums for differentiation

in to neuronal cells for specific time internal. • Caffeine, decaffeinated, Tobacco and Quest drugs are

added at 3DIV.• Immunocytochemistry• Analysis: take images of all LB like aggregations and

DA cells.• The size of average LB size for each batch is obtained• The ratio of DA cells/LB is obtained for each

treatment group.

Results for control at 1,2,3,6 and 9DIV

• Most significant figure in this chart is the average size of LB over time remains stable starting from 2DIV.

2 3 6 90.000

2.000

4.000

6.000

8.000

10.000

12.000

14.000

Control: Average LB and DA cell Sizes

Ave LB Size

Ave DA Cell Size

Days In Vitro

Siz

e (

μm

)

N=34 N=61 N=21 N=18

• Provided that the sizes of LB over time is relatively stable from 2 to 9DIV, the ratio of total number of LB cells versus #DA is also stable. However, the ratio for 1DIV is 3 times higher. This information tells me that the formation of LB is between 1DIV and 2DIV.

1 2 3 6 90.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

4.000

4.500

5.000

0.077

3.548

2.799

3.974

3.317

Control total #LB/DA Ratio

Days In Vitro

Tota

l #LB

/DA

ratio

Coffee and Decaff extract added at 3DIV• The addition

of coffee and decaff extracts at 3DIV reduces the number of LB in both cases.

• However, the ratio declines either because of an increase of LB as the drugs are used up over time or a decrease in total number of DA cells.

6 90.000

2.000

4.000

6.000

8.000

10.000

12.000

14.000

Chart Title

ControlCoffeeDecaff

Days in Vitro

Tota

l #

LB

/DA

Rati

o

N=769/24

N=823/102N=615/72

N=276/50

Conclusion:• Lewy Body is filamentous protein aggregation with a

size range of 4 to 7μm which mainly constitutes α-synuclein and it is up regulated early in drosophila neuronal cultures.

• From these experiments, the formation of LB is studied in both the absence and presence of neuroprotective drugs.

• The number of LB increases drastically between 1DIV and 2DIV and stays stable both in numbers and sizes.

• The data shows that both coffee and decaff extracts reduce the number of LB after LBs are fully formed which implies that these drugs are involved in the degradation of LBs.

References:• Auluck et al 2010• Mel B. Feany and Welcome W. Bender. A drosophila model of Parkinson’s disease• Pavan K. Auluck, Gabriela Caraveo and Susan Lindquist. Α-synuclein: Membran Interactions and Toxicity In

Parkinson’s Disease.• Walter J. Schulz-Schaeffer. The synaptic Pathology of α-synuclein aggregation in dementia with Lewy bodies,

Parkinson’s disease and Parkinson’s disease dementia.